The detection and characterization of certain types of biological cells has proven useful in identifying and discriminating between certain disease states. To date, such interrogations have been performed by three classes of instruments, those employing standard microscopy techniques, those utilizing pattern analysis techniques, and those based on flow cytometry technology.
Standard microscopy is the oldest of these techniques and conceptually the simplest to understand. Standard microscopy optically examines, by means of a series of glass lenses in the objective and ocular portions of a microscope, cells placed on the surface of a transparent support surface or slide. A field encompassing the cells of interest is exposed to visible or ultraviolet light and the cells' optical absorption or fluorescence is observed or manually measured using a photodetector. Signal changes can then be related to altered levels of cellular constituents such as proteins or nucleic acids. These procedures tend to be tedious, time consuming and are difficult to apply to living cells. Hence, although useful for obtaining morphological information, they are inappropriate for deriving dynamic or time varying information from biological cells. It is an object of the present invention to overcome these inherent, standard microscopy limitations.
Subsequently, pattern analysis techniques were developed to provide the capability to scan, en masse, a plurality of conventionally stained cells which have been flattened out on slides and to process the resulting data. As a result, these techniques have permitted the development of a class of clinical laboratory instruments which have automated certain manual procedures previously employed in standard microscopy. Pattern analysis has, however, because of the staining techniques been confined to measuring morphological properties of nonviable cells rather than biophysical properties of live cells and their constituents. Hence, its usefulness has been limited to differentiating cell types in heterogeneous populations.
It is an object of the present invention to provide a new class of apparatus and methods utilizing same for measuring individual, kinetic or time varying biophysical properties of live cells and cellular constituents; properties which cannot be determined with present pattern analysis techniques.
Still another well-known class of clinical and research laboratory, light based instruments include those based on the so-called flow cytometry technology. Typically, these instruments hydrodynamically focus fluid suspended cells into a single file stream for passage through an examination zone. While in the zone, the cells interact with impinging light from a focused light source, ideally a laser. One or more optical interactions of the cells and the light are measured and may include, for instance, multiple wavelength absorption, scatter as a function of angle, fluorescence as a function of wavelength, polarization, and the like. This class of instruments permits the study of living cells in addition to those which have been chemically treated. Flow cytometry techniques also enable certain constituents or structures, particularly those present on the cell surface, to be quantitatively characterized at cell rates of a thousand or more cells per second. With such great sampling rates, the size, shape and number of large populations of cells may be readily studied thereby permitting the biologist to gain statistically significant information about the numerically small subpopulations cf cells comprising the heterogeneous population.
Flow cytometry techniques, in addition to the previously described techniques, however, commonly suffer from an inability to practically measure the kinetic properties of live cells in large heterogeneous populations. They are also unsuitable for the practical study of the flow of biological information in and between living cells such as neurons, blood cells and the like. This inability stems predominantly from the instruments' incapacity to maintain the identity of each cell measured and/or the instruments' inability to perform the measurements over a cellularly significant passage of time in the short of time interval during which the cell is present in the examination zone.
It is an object of the present invention to provide means and methods for studying the kinetic properties of live cells in large heterogeneous populations.
It is a related object of the present invention to provide means whereby the exchange of biological information inter and intra-cellularly may be measured.
It is a further object of the present invention to provide means and methods which do not employ instruments of the classes previously discussed.
It is a related object of the present invention to provide an instrument which relies upon the sensitivity provided by cells in order to detect the presence or absence of materials in a fluid brought into contact with the cells and thereby also provide improved sensitivity in the detection of such materials.
It is a yet further object of the present invention to provide an apparatus capable of detecting specific types of cells which may be differentiated by their reactivity with and sensitivity to reagents brought into contact with those cells.
It is a still further object to provide apparatus and methods capable of performing cellular indentification and measurements in the requisite time frames to obtain the kinetic information desired.